NF-κB p65 (D14E12) XP® Rabbit mAb (Cell Signaling Technology, #8242)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Chromatin Immunoprecipitation (ChIP) was performed as described (Luo, et al. Mol Cell). FIP cells from control or Mural-Zfp423TG mice were cross-linked with 1% formaldehyde in PBS for 10 minutes at 37 °C and quenched in 125 mM glycine in PBS for 5 minutes at 4 °C. The FIP cells were then lysed in Farnham lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 1 mM DTT, and Protease inhibitor cocktail [Sigma, #P8340]) to obtain nuclear material. Crude nuclear pellets were collected by centrifugation and resuspended in lysis buffer (5 mM Tris-HCl pH 7.9, 1% SDS, 10 mM EDTA, 1mM DTT, and Protease inhibitor cocktail [Sigma, #P8340]), and incubated on ice for 10 minutes. The chromatin fragmentation was performed at 4 °C by Bioruptor 300 using the setting of 10 cycles of 30 seconds on and 60 seconds off to generate chromatin fragments of 200-500 bp in length. The soluble chromatin fragments were diluted 1:10 with dilution buffer (20 mM Tris-HCl pH 7.9, 0.5% Triton X-100, 2mM EDTA, 150 mM NaCl, 1 mM DTT, and Protease inhibitor cocktail [Sigma, #P8340]) and pre-cleared using Protein G Sepharose 4 Fast Flow (GE Healthcare Bio-sciences, #17-0618-01) for 1 hour at 4 °C. The pre-cleared samples were incubated with anti-p65 antibody (Cell Signaling technology, #8242) at 4 °C for overnight before the addition of Protein G Sepharose 4 Fast Flow (GE Healthcare Bio-sciences, #17-0618-01) at 4 °C for 2 hours to capture the antibody-protein-DNA complexes. The immunoprecipitated material was consecutively washed with low salt wash buffer (20 mM Tris-HCl pH 7.9, 2 mM EDTA, 125 mM NaCl, 0.05% SDS, 1% Triton X-100, and Protease inhibitor cocktail [Sigma, #P8340]), high salt wash buffer (20 mM Tris-HCl pH 7.9, 2mM EDTA, 500 mM NaCl, 0.05% SDS, 1% Triton X-100, and Protease inhibitor cocktail [Sigma, #P8340]), LiCl wash buffer (10 mM Tris-HCl pH 7.9, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, and Protease inhibitor cocktail [Sigma, #P8340]), and 1x Tris-EDTA (TE). After eluted using elution buffer (100 mM NaHCO3, 1% SDS), the immunoprecipitated material was digested with RNase (Roche, #11119915001) and proteinase K (ThermoFisher Scientific, #EO0491) prior to the purification and concentration of the immunoprecipitated genomic DNA by ChIP DNA Clean & Concentrator kit (Zymo Research, #D5201). ChIP-seq libraries were generated using Nebnext NGS DNA Library Preparation for Illumina kit (New England BioLabs, E7645) following the manufacturer's protocol.